Abstract Post-transplant lymphoproliferative disorders (PTLDs) remain a feared malignant complication of transplantation, with high 5-year mortality and morbidity exceeding 50%. About 50-80% of cases are strongly related to the oncogenic Epstein-Barr virus (EBV), a key pathogenic driver. Many knowledge gaps exist in PTLD. Several prognostic indices, comprising multiple clinical, epidemiological and tumor characteristics, including EBV tumor positivity, do not consistently associate with worse patient survival, suggesting an additional role for EBV genome variants or other viral etiologies. However, the precision medicine tools for determining if a viral genome variant is pathogenic are very limited compared to human genome variants. Further, the etiological agent in EBV-negative PTLD is unknown. Using novel recently developed cutting-edge technologies, we can extract viral nucleic acids from formalin-fixed, paraffin-embedded archived PTLD tissues or plasma and sequence multiple viruses simultaneously in unbiased fashion, using metagenomic shotgun sequencing (MSS) and ViroCap?. Based on our preliminary data, we propose a precision medicine translational genomics project to address the following specific aims and close the cited knowledge gaps: 1) Validate our novel observation that PTLD tissue positivity by MSS for anellovirus (and confirmed by other techniques) serves as a biomarker for higher transplant recipient mortality after the diagnosis of PTLD; 2) Determine the role of other oncogenic viruses in EBV-negative PTLD by unbiased MSS of multiple viral groupings, confirmed by other techniques; 3) Develop the necessary computational, algorithmic and software analytic tools required to then determine association of EBV genome variants with worse presentations or outcomes in PTLD. To achieve these aims, we will retrospectively collect PTLD tissues and prospectively collect PTLD tissues/plasma from: 1) cases at Washington University that have occurred after 2015; 2) three USA sites with large transplant programs and existing PTLD collections (Universities of Pennsylvania, Pittsburgh and Stanford; 3) a French national-level PTLD registry that already has extensive clinical data and can obtain archived tissues. In this proposed sample size of adequately powered 630 PTLD cases, we will: a) acquire tissue scrolls from the PTLD samples and transport to Washington University; b) extract the microbial DNA and RNA; c) perform metagenomic shotgun sequencing; and d) validate our novel associations to the clinical data, imported from each of the sites, into an already constructed RedCap database. Our team includes specialists in transplantation, hematopathology, infectious diseases, oncology, genomics, data management and statistics. Our preliminary data, collaborators and local resources are exceptional to accomplish these goals. This study will additionally create the largest-to-date repository of combined PTLD tissue, extracted nucleic acids, and well-annotated clinical and pathological data, for use in further research. Completion of this study will contribute to better patient care and may provide avenues for novel therapies.